Complement 1s is the serine protease that cleaves IGFBP-5 in human osteoarthritic joint fluid.
Insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) are trophic factors for cartilage and have been shown to be chondroprotective in animal models of osteoarthritis (OA). IGFBP-5 is degraded in joint fluid and inhibition of IGFBP-5 degradation has been shown to enhance the trophic effects of IGF-I.


OBJECTIVE
To determine the identity of IGFBP-5 protease activity in human OA joint fluid.


METHODS
OA joint fluid was purified and the purified material was analyzed by IGFBP-5 zymography.


RESULTS
Both crude joint fluid and purified material contained a single band of proteolytic activity that cleaved IGFBP-5. Immunoblotting of joint fluid for complement 1s (C1s) showed a band that had the same Mr estimate, e.g., 88 kDa. In gel tryptic digestion and subsequent peptide analysis by LC-MS/MS showed that the band contained human C1s. A panel of protease inhibitors was tested for their ability to inhibit IGFBP-5 cleavage by the purified protease. Three serine protease inhibitors, FUT175 and CP-143217 and CB-349547 had IC50's between 1 and 6 microM. Two other serine protease inhibitors had intermediate activity (e.g., IC50's 20-40 microM) and MMP inhibitors had no detectible activity at concentrations up to 300 microM.


CONCLUSIONS
Human OA fluid contains a serine protease that cleaves IGFBP-5. Zymography, immunoblotting and LC-MS/MS analysis indicate that C1s is the protease that accounts for this activity.
Busby
W H
WH
Department of Medicine, University of North Carolina, School of Medicine, Chapel Hill, NC 27599, USA.
Yocum
S A
SA
Rowland
M
M
Kellner
D
D
Lazerwith
S
S
Sverdrup
F
F
Yates
M
M
Radabaugh
M
M
Clemmons
D R
DR
D003173:Complement C1s / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism*; D020370:Osteoarthritis, Knee / Q000201:enzymology / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D057057:Serine Proteases / Q000378:metabolism / Q000502:physiology*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D013582:Synovial Fluid / Q000378:metabolism*
D018975:Insulin-Like Growth Factor Binding Protein 5; D010446:Peptide Fragments; D015842:Serine Proteinase Inhibitors; D057057:Serine Proteases; D003173:Complement C1s